1. Volume 12, Issue 5, Pages 900-909 (November 2005)
Long-Term Inhibition of HIV-1 Infection in Primary Hematopoietic Cells by Lentiviral Vector Delivery of a Triple Combination of Anti-HIV shRNA, Anti-CCR5 Ribozyme, and a Nucleolar-Localizing TAR Decoy 
Ming-Jie Li, James Kim, Shirley Li, John Zaia, Jiing-Kuan Yee, Joseph Anderson, Ramesh Akkina, John J. Rossi 
Molecular Therapy 
Volume 12, Issue 5, Pages (November 2005)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
(A) The lentiviral vectors. The lentiviral vector backbone pHIV-7-GFP contains a hybrid 5′ LTR in which the U3 region is replaced with the CMV promoter, the packaging signal (Ψ), the RRE sequence, the flap sequence, the woodchuck posttranscriptional regulatory element (WPRE), and the 3′ LTR in which the cis regulatory sequences are completely removed from the U3 region. The genes of interest along with their independent Pol III promoters are inserted upstream of the CMV promoter of EGFP in the pHIV-7-GFP vector. U6, U6 promoter; shI, shRNA against a common rev/tat exon; TAR, U16-TAR RNA decoy; VA1, VA1 promoter; RZ, anti-CCR5 ribozyme. Arrows indicate the transcriptional orientation. (B–D) The therapeutic genes and their targets. (B) The expression cassette of the shRNA targeting rev/tat is composed of the U6 Pol III promoter, the sense and antisense sequences of the shRNA separated by a 9-base loop, and a terminator with 6 thymidines. The putative shRNA after transcription in target cells is shown. (C) The TAR RNA decoy in the U16 snoRNA. The stem–bulge–loop configuration of the TAR decoy is shown at the top. The TAR decoy is inserted into the apical stem of the U16 snoRNA sequence, allowing its nucleolar localization [31]. C box and D box are the U16 nucleolar localization signals. (D) The anti-CCR5 hammerhead ribozyme and the CCR5 sequence complementary to the ribozyme. The ribozyme binds to the substrate with its two arms and cleaves immediately 3′ of the GUC codon as indicated by the arrow. The ribozyme is inserted within the VA1 coding region as previously described [28].
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Titers of lentiviral vectors with various inserts. The vectors were produced by cotransfection of 293 T cells with all required plasmids, among which pCMV-rev was at either 5 (solid bars) or 10 μg (hatched bars). HT1080 cells were transduced with various packaged vectors as indicated. Forty-eight hours after transduction, the cells were analyzed by FACS for EGFP expression. The titers were represented as the number of EGFP+ cells per milliliter of concentrated vectors. The values are averages from three independent experiments.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Expression of the therapeutic RNAs in the target cells. (A) CEM cells and CD34+ cells were transduced with the lentiviral vectors harboring the expression cassettes for the anti-tat/rev shRNA (shI), U16TAR decoy, and anti-CCR5 ribozyme. The CD34+ cells were sorted for EGFP+ cells. Two weeks after transduction (for CEM cells) or sorting (for CD34+ cells), total RNA was extracted from the CEM cells and the EGFP+ population of the CD34+ cells, electrophoresed on an 8% polyacrylamide gel with 7 M urea, blotted onto a nylon membrane, and hybridized with a 32P-labeled probe for the antisense sequence of the shI or mixed probes for the U16TAR decoy and anti-CCR5 ribozyme. A U6 snRNA complementary probe was used as an internal control. RNA extracts from untransduced cells and cells transduced with the EGFP parental vector or a vector expressing an unrelated shRNA were used as negative controls. The amounts of RNA loaded into the gel were 15 μg for CEM cells and 7.5 μg for CD34+ cells. (B) CD34+ cells were transduced with the vectors at the indicated m.o.i. After sorting, EGFP+ cells were cultured for 9 weeks. The conditions for RNA extraction and Northern blotting were the same as in (A), except that the amount of RNA was 1.5 μg in each sample.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Transduction efficiency of the vectors in CD34+ cells. CD34+ cells were transduced with various lentiviral vectors at an m.o.i. of 40. Eleven days after transduction, FACS analyses were performed to detect EGFP+ cells. The x axis indicates the intensity of fluorescence. The y axis indicates the number of cells.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Inhibition of HIV-1 replication in CD34+ cells expressing the anti-rev/tat shRNA, TAR decoy, and anti-CCR5 ribozyme. The CD34+ cells were transduced with the vectors indicated. The EGFP-expressing cells were collected by FACS and infected with HIV-1 strain JR-FL at m.o.i. of (A) 0.01 and (B) The culture supernatants were collected weekly up to 4 weeks. The concentration of HIV-encoded p24 antigen was determined by ELISA.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Detection of HIV-1 in CD34+ cell culture following long-term viral infection. (A) Detection of HIV-1 p24 antigen. CD34+ cells were transduced with the vector expressing the triple therapeutic genes or the parental vector pHIV-7 at an m.o.i. of 10. After sorting, EGFP-expressing cells were collected by FACS and infected with the HIV-1 strain JR-FL at an m.o.i. of The culture supernatants were collected at the days indicated and the concentration of HIV-encoded p24 antigen was determined by ELISA. (B) Detection of HIV-1 viral genomes in culture supernatants. Viral RNA was extracted from the culture supernatants from the experiment depicted in (A) at the selected time points. The RNA was amplified by RT-PCR with primers flanking the anti-rev/tat shRNA target sequence. (C) Detection of HIV-1 in CD34+ cells after rechallenge. Untransduced CD34+ cells were challenged with the day 28 supernatants collected from the previous challenge. The supernatants from the rechallenged cultures were collected up to 4 weeks and the concentration of HIV-1 p24 antigen was determined by ELISA.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

8. FIG. 7
(A) B7.1 up-regulation of LPS-stimulated CD34+-derived macrophages. Nontransduced, HIV-7-transduced, and triple construct lentiviral vector-transduced macrophages were stimulated with LPS. Twenty-four hours poststimulation, cells were stained with PE-Cy5-conjugated anti-B7.1 antibody and analyzed by FACS. These data are representative of triplicate experiments. (B) Phagocytosis of fluorescently labeled Escherichia coli by CD34+-derived macrophages. E. coli Bioparticles were added directly to the cultured cells. Twenty-four hours later, cells were analyzed by FACS. Graph A, control macrophages without Bioparticles. Graphs B–E show cells incubated with E. coli Bioparticles. Graphs B, transduced Magi-CXCR4 (nonphagocytic cell control); C, nontransduced; D, HIV-7 transduced; and E, triple construct lentiviral vector-transduced macrophages. Labeled E. coli Bioparticles were analyzed in the PE channel. These are representative of triplicate experiments.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions